This invention relates to biotechnology generally, and more specifically to an ex vivo animal or challenge model as a method to measure protective circuitry directed against parasites and vaccines shown to be protective in the method.
Only a few vaccines against parasites are commercially available. Most of these vaccines are based on attenuated live parasites that induce natural, protective immunity and cause less severe pathological damage. These parasite vaccines include one directed against Dictyocaulus viviparus (e.g., Dictol, Glaxo), undoubtedly the most successful anti-parasite vaccine, and analogous therewith a vaccine against Dictyocaulus filaria, the lung worm in sheep (Sharma et al. 1988). These vaccines are based on live but irradiated third-stage larvae (Peacock and Pointer 1980). Another attenuated vaccine is directed against the hookworm Ancylostoma caninum in dogs. However, this vaccine has been marketed only for a short time in the USA, marketing was discontinued because the American veterinary profession did not accept this live vaccine (Urquhart 1980). An attenuated vaccine against Babesia bovis has been in use for nearly a century in Australia (Purnell 1980) and a dead vaccine based on metabolic products named xe2x80x9cPirodogxe2x80x9d is used to vaccinate dogs against B.canis (Moreau 1986).
Vaccination trials in sheep with a recombinant vaccine against the tape worm Taenia ovis (Johnson et al. 1989) and the concealed antigen H11 from Haemonchus contortus (Newton 1995, review) have been performed successfully. A trial with the SPf66 malaria vaccine in Africa has recently been completed. The efficiency against clinical malaria in areas of high transmission was 31% and the product appeared to be safe. However, because it is not fully understood how SPf66 mediates protection, the development of improved vaccines is hampered (Tanner et al. 1995; review).
Problems of developing anti-parasite vaccines are abundant. Parasites have complex life cycles and each stage expresses different sets of antigens. Moreover, the different stages are often associated with different sites in the body. For most parasites little is known about the immune mechanisms involved in natural immunity and about the stage of the parasite inducing this immunity.
Most often, no reproducible animal model is available to study these mechanisms, thereby blocking a new approach in vaccine development. As mentioned above, most available vaccines are based on attenuated live parasites. These vaccines can sometimes be successful because the xe2x80x9cvaccine parasitesxe2x80x9d follow the correct route of infection and deliver a wide array of stage-specific antigens. However, such vaccines must challenge the acceptance of the public (e.g., Ancylostoma caninum vaccine), especially when they are for human use (e.g., Schistosoma mansoni vaccine, Taylor et al. 1986). Moreover, live vaccines, in general, have a short shelf-life and are relatively expensive. From this perspective there is an obvious need for vaccines that are based on (recombinant) proteins derived from the parasite. However, the identification of such protective proteins meets a great number of difficulties, as shown below as an example for Fasciola hepatica. 
The trematode parasite Fasciola hepatica mainly infects cattle and sheep. Sometimes also humans get infected. The parasite causes considerable economic losses in, for example, western Europe, Australia and South America. The metacercariae of Fasciola hepatica enter its host by the oral route, penetrate the gut wall within 4-7 hours (Dawes 1963, Burden et al. 1981, Burden et al. 1983, Kawano et al 1992) and migrate through the peritoneal cavity towards the target organ, the liver. Oral infection of cattle results in almost complete protection against a challenge, whereas sheep often die from an infection and do not acquire natural immunity. Both the natural host (cattle) and the animal model (rat) acquire natural immunity after infection (Doy and Hughes 1984; Hayes, Bailer and Mitrovic 1973). Therefore, rats are often used to study resistance in cattle. In the rat a large part of natural immunity is expressed in the gut mucosa, the porte d""entree of the parasite. In immune rats, about 80% of the challenge newly excysted juvenile stages (NEJs) is eliminated in the route from the gut lumen to the peritoneal cavity (Hayes and Mitrovic 1977, Rajasekariah and Howell 1977, Doy, Hughes and Harness 1978/1981, Doy and Hughes 1982, Burden et al. 1981/1983). Based on natural immunity, a vaccine based on irradiated Fasciola gigantica metacercariae was developed for cattle (Bitakaramire 1973). In the seventies and eighties many vaccination experiments have been performed with antigen extracts of adult and juvenile flukes (Haroun and Hillyer 1980, review). However, these studies lead to conflicting or disputable results. For example, subcutaneous or intramuscular injection of rats with adult or juvenile fluke extracts did not result in protection (Oldham and Hughes 1982, Burden et al. 1982, Oldham 1983). Adult fluke extracts given intraperitoneally in Freund complete adjuvant (FCA) or incomplete Freund adjuvant (IFA) resulted in about 50% protection (Oldham and Hughes 1982, Oldham 1983). Using very high antigen doses of Bordetella pertussis as additional adjuvant this protection reached 80-86% (Oldham and Hughes 1982, Oldham 1983). Extracts of 4-week-old juveniles given intraperitoneally in AIOH3 did not induce protection in the studies of Pfister et al. (1984/85), whereas 16-day old juvenile extracts provided 86% protection in mice, without the use of adjuvant (Lang and Hall 1977). Subcutaneous sensitization of cattle with sonicated 16-day-old juveniles resulted in more than 90% protection (Hall and Lang 1978). Intramuscular injection of calves with an isolated fraction from adult Fasciola hepatica (FhSmIII), with an immunogenic 12 kD protein as major component, resulted in 55% protection (Hillyer et al. 1987).
Since 1990, several Fasciola hepatica vaccine candidate antigens have been isolated and/or produced. Most of these antigens are derived from adult flukes and share homology with Schistosoma mansoni antigens. Glutathion S-transferases (GST) are enzymes amongst others active in the cellular detoxification system. Immunization of sheep (n=9) with GST purified from adult Fasciola hepatica, injected s.c. in FCA, with a boost immunization 4 weeks later in IFA, resulted in 57% protection (Sexton et al 1990). Immunization of rats with GST provided no protection (Howell et al. 1988). Vaccination trials in cattle performed by Ciba Animal Health Research (Switzerland) and The Victorian Institute of Animal Science (Australia), resulted in 49-69% protection (Morrison et al. 1996).
Intradermal/subcutaneous immunization with recombinant S.mansoni fatty acid-binding protein Sm14 in FCA, provided complete protection against Fasciola hepatica challenge in mice (Tendler et al. 1996). PCT International Patent Publication WO 94/09142 suggests the use of proteases having cathepsin L type activity, derived of Fasciola hepatica, in the formulation of vaccines for combatting helminth parasites; immunisation of rabbits with the purified mature enzyme resulted in rabbit antibodies capable of decreasing the activity of the enzyme in vitro.
However, levels of protection obtained with F. hepatica cathepsin L or haemoglobin in cattle were only 53.7% or 43.5%, respectively (Dalton et al. 1996). Cathepsin L belongs to a family of cysteine proteinases, secreted by all stages of the developing parasite. Cathepsin L from F. hepatica is most active at slightly acid or neutral pH (Dalton and Heffernan, 1989). The functions of this proteinase include disruption of host immune function by cleaving host immunoglobulin in a papain-like manner (Smith et al. 1993) and preventing antibody mediated attachment of immune effector cells to the parasite (Carmona et al. 1993). Moreover, cathepsin L is capable of degradation of extracellular matrix and basement membrane components (Berasain et al. 1997), and prepares mucosal surface to be penetrated by a parasite indicating that cathepsin L is involved in tissue invasion. Because of its crucial biological functions, cathepsin L proteases are considered important candidates for the development of an anti-parasite vaccine.
Cathepsin L is synthesized as a preproprotein with a 15 amino acid (xe2x80x9caaxe2x80x9d) long peptide presequence, a 91 aa long peptide prosequence or proregion and a 220 aa long (poly)peptide enzymatic part. Of cysteine proteinases the preregion is removed immediately after synthesis and the proprotein comprising the proregion and the part that (constitutes the mature enzyme) is transported to the Golgi. Conversion to the mature enzyme and thus conversion to an enzymatically active state, occurs in the lysosomes and could be due to cathepsin D or to autoactivation. In some cases precursors containing the proregion are secreted (North et al. 1990). Cathepsin L itself has a high affinity for a substrate with Arg at the P1 position and a hydrophobic residue (Phe) at the P2 position (Dowd et al. 1994). It also has autocatalytic activity and cleaves off its prosequence before it obtains its mature enzymatic activity. Cathepsin L2 also cleaves peptides containing Pro at the P2 position, and is therefore capable of cleaving fibrinogen and producing a fibrin clot.
Other potential candidates for an anti-fluke vaccine are hemoglobin, isolated from mature Fasciola hepatica (McGonigle and Dalton 1995) and cathepsin L secreted by adult Fasciola hepatica (Smith et al. 1993; Smith et al. 1994; Spithill 1995). Up to now, next to the irradiated Fasciola gigantica metacercariae (Bitakarami 1973) several antigens have been named as potential protein vaccines:
Fasciola hepatica haemoprotein
Fatty acid-binding protein Sm14 from Schistosoma mansoni 
Thiol proteases with Cathepsin L-type activity
Glutathion S-tranferase extracted from adult Fasciola hepatica 
polypeptide from Fasciola species (Gln-Xaa5-Cys-Trp-Xaa3)
Serin proteases with dipeptidyl peptidase activity
However, none of these potential candidates have emerged as an effective vaccine against Fasciola hepatica infection, and a large number of questions, such as: at what site in the host is immunity expressed?; against which stage of the parasite is immunity directed?; at which site in the host this immunity is induced?; which immune mechanisms are involved in protection?; which stage of Fasciola hepatica induces protective immunity?; andxe2x80x94last but not leastxe2x80x94which antigens induce protection?, need to be answered before a successful vaccine can be developed. It is clear that answering these questions is greatly hampered by the lack of a suitable animalxe2x80x94or challenge model by which parasitic infections can be studied. And even when animal models are available progress can only be slow because of the fact that the parasitic infection in the host under study takes a considerable time to develop while its outcome depends on various factors that relate to the in time changing host-parasite relationship. For instance, although much focus has been directed to proteins, such as proteases, derived from newly excysted juvenile (NEJ) stages of Fasciola hepatica as candidate protective antigens (see for instance Tkalcevic et al, 1995), no clear cut identification of truly protective proteins has been foreseen. To the contrary, early developmental stages of Fasciola hepatica display rapid changes in protein and antigen expression during the early stages of infection, and such changes may even assist the parasite to evade the host immune response (Tkalcevic et al, Parasite Immunology 18: 139-147, 1996). It has for instance been demonstrated that in parasites, proteases are involved in the invasion of host tissues, the evasion of immune attack mechanisms and help provide nutrients for parasite survival.
Thus, both the abundance of possible different proteins or antigens that need to be studied and the lack of suitable challenge models hamper the possible progress that is needed in the development of parasite vaccines. Crucial for progress in parasite vaccines are new methods to measure protective immunity in order to be able to study a variety of candidate protective antigens and to identify new candidate protective antigens. Thus new animal models are needed that will increase the number of candidate proteins or substances that can be tested in time.
The present invention provides a very rapid method to study, investigate and evaluate natural immunity against a parasite under study. The invention provides an ex vivo animal or challenge model method to rapidly study protective immunity directed against parasites and vaccines directed against parasitic infections. Ex vivo models are in general designed to study organs or organsystems of animals, under anaesthesia, out of the context provided by the natural body, but still within the context of proper blood supply or the like. These models have in general a short execution time and provide less prolonged suffering to the experimental animal than seen with in vivo models.
The invention provides an ex vivo gut model in the rat, or in other small experimental animals such as mice or chickens, or in other animal species. Challenge parasites are injected in one or more ex vivo segments of the intestines of the selected animal and parasites, such as NEJs, that than penetrate the intestinal wall are recovered in a container that holds the particular gut segment. In particular, segments of the small intestine, such as duodenum, jejunum or ileum can be used, however, segments of other parts of the intestine, such as stomach, colon, caecum or rectum can also be used depending on the selected route of infection of the parasite under study. This model provided by the invention is capable of measuring expression of resistance in the entire intestine by comparing segments that have been subjected to different loads of parasites or to different stages of parasites. In addition, all the trajects in the migration route of the parasite such as be can found in gut mucosa, peritoneal cavity and liver and others, which are essential for the induction of mucosal resistance can be investigated. Such studies that are enabled by the invention provide knowledge about the most efficient vaccination route and about possibilities for an oral vaccine. Another advantage of the ex vivo challenge model using ligated gut segments is that migration of the pathogen from the gut lumen to the peritoneal cavity is limited to a small area, allowing the localisation and characterization of the protective immune response against the parasite in the gut mucosa. Moreover, the level of resistance induced by a previous infection or vaccination can be correlated with immune mechanisms against the parasite (in the experimental part demonstrated with Fasciola hepatica) because the challenge infection does not settle and does not induce additional immune responses that interfere with those that need to be studied. Especially the immunity and protective mechanisms directed against those pathogens that penetrate mucosal or skin surfaces to infect the host, such as Fasciola hepatica, Paragonimus westermani, Schistosoma mansoni, Toxocara canis, Dictyocaulus viviparus, Trichinella spiralis, Nematodiris spp, Nippostrongylus brasiliensis, Ascaris suum, Anisakis and other pathogens varying from prions to protozoa, whether they may fully or partly penetrate said surfaces, can be measured specifically well by the model provided by the invention. Parasites or other pathogens that fully penetrate the mucosal surfaces of the gut segments employed in the model can be recovered as shown above, those that only partly penetrate the mucosal surfaces can be recovered from the bloodxe2x80x94or lymphvessels servicing the particular segment.
Measuring the immunity and protective mechanisms directed against parasites offers the possibility to modulate the effector phase of the immune response in the host which will result in the development of efficient vaccination strategies. In other words, the invention measures the capacity of proteins to be protective antigens for use as vaccine against infections. Because protection data are obtained the same day the ex vivo model provided by the invention enables quick testing of different stages of many candidate vaccine antigens (protective proteins or fragments derived thereof) for their capacity to induce resistance and immunity.
One such candidate vaccine antigen provided by the invention is a protective protein, or antigenic fragment derived thereof, said protein at least comprising an amino acid sequence derived of a proregion of an enzyme. Several proteases are involved when a parasite penatrates a mucosal or skin surface. Examples are serine protease, dipeptidyl peptidase-like protease, cysteine protease, proteases with cathepsin-like activity, but also enzymes like glutathion S-transferase and many others are involved during the phase when the parasite is penetrating a mucosal or skin surface. Surprisingly, the invention provides protective protein (fragments) derived of a proprotein of such an enzyme or protease which elicit a better immune response than when a mature enzyme is used. Optionally, it is possible to combine the immune response directed against the proprotein with the immune response directed against the mature enzyme.